Autism is a disorder defined by both American and International diagnostic systems (i.e., the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) and World Health Organization: International Classification of Diseases, Tenth revision (ICD-10)). Autism is the third most prevalent developmental disability in the United States, currently affecting over one-half million people. The disorder is typically characterized by multiple distortions in the development of basic psychological functions that are involved in the development of social skills and verbal and non-verbal communication, such as attention, perception reality testing and motor movement. Children and adults suffering from autism may exhibit repeated body movements, unusual responses to people or attachments to objects and may resist any changes in routines. In some cases, those suffering from the disorder may exhibit aggressive or self-injurious behavior. Additionally, many patients diagnosed with Autism suffer from primary diffuse gastrointestinal problems such as protracted diarrhea and constipation. The specific cause of Autism is not known and there is no known cure for the disorder. Additionally, conventional methods of treatment, including dietary alteration, behavioral modification, and medication, have proven unsuccessful in allowing such children and adults to become symptom, or disorder free.
To date, there is no comprehensive treatment for the broad range of autistic symptomatology: seizures (Park 2003); attentional/arousal dysregulation, ADHD (Booth, et al., 2003); obsessive-compulsive disorder (OCD) (Hollander, et al., 2003a); stereotypies (Militemi, et al., 2002); social isolation (Iqbal 2002); attachment disorders (Tinbergen and Tinbergen 1983, Kobayashi, et al., 2001); face recognition deficits (Ogai, et al., 2003, Schultz, et al., 2003); gaze aversions (Richer, et al., 1976); gastrointestinal disorders (Horvath, et al., 1998, Horvath, et al., 2002, Torrente, et al., 2002, Gershon 2003 personal communication); and altered heart rate variability (Graveling and Brooke 1978, Corona, et al., 1998). Current drugs directed at treating these symptoms have long-term side effects and efficacy not far above placebo rates (Posey, et al., 2000), resulting in motivation to seek new treatments.
Gastrointestinal (GI) disorders, including inflammatory bowel diseases (IBDs), affect millions of people of all ages world wide, and the social and economic costs of these disorders are enormous. The symptoms of GI disorders range from inconvenience and mild discomfort to total incapacitation. For those with severe symptoms, GI disorders can be debilitating, rendering participation in social and professional activities impossible. Because much remains unknown about GI disorders, misdiagnosis and ineffective treatment for these disorders is common. For example, women suffering from irritable bowel syndrome (IBS) have an increased risk of unnecessary surgery including unnecessary hysterectomy and ovarian surgery. Longstreth G F, “Irritable Bowel Syndrome: A Multibillion-Dollar Problem” Gastroenterology, 109:2029-2042 (1995). Accordingly, a current need exists for new and better methods for improving the prognosis of patients suffering from GI disorders.
The pathogenesis of inflammatory bowel diseases (IBDs) is multifactorial, involving immune dysfunctions, specifically a dysregulation of Th1/Th2 type cytokines (Iijima, et al., 1999). Alteration of interleukin 4 production results in the inhibition of T helper type 2 cell-dominated inflammatory bowel disease in T cell receptor alpha chain-deficient mice. J. Exp. Med., 190(5): 607-15; Dohi, et al. Hapten-induced colitis is associated with colonic patch hypertrophy and T helper cell 2-type responses. J. Exp. Med., 1999; 189(8): 1169-80; Kucharzik, et al., Synergistic effect of immunoregulatory cytokines on peripheral blood monocytes from patients with inflammatory bowel disease. Dig. Dis. Sci., 1997, 42(4):805-12). Such Th1/Th2 dysregulation has been suggested in the pathogenesis of autism as well. (Gupta, et al., 1998, 85(1):106-9.) Interestingly, a shift from Th1 to predominantly Th2 cytokines is induced by vasoactive intestinal peptide (VIP) (Dohi, et al.). Elevated levels of plasma VIP in neonates later diagnosed with autism were reported in a large prospective study (Dohi, et al.). Some cases of autism are reported with onset subsequent to an IBD episode. (Lightdale, et al., Gastronintestinal symptoms in autistic children, Clin. Perspec. Gastroenterol., 156-58 (2001); White, Intestinal pathophysiolgy in autism, Exp. Biol. Med. (Maywood) (2003) 228(6):639-49; Horvath, et al., 1999, Gastronintestinal abnormalities in children with autistic disorder; J. Pediatr. 135(3):559-63.) Autism is also associated with a high incidence of familial autoimmune disorders (Comi, et al., Familial clusterin of autoimmune disorders and evaluation of medical risk factors in autism, J. Child. Neurol., 1999, 14(6):388-94) and is often associated with a familial finding of elevated plasma levels of serotonin (5HT) (Cook, Autism: review of neurochemical investigation. (Synapse, 1990, 6(3):292-308). The body's predominant source of 5HT is the gastrointestinal tract. (Gershon 1998). The Second Brain: a groundbreaking new understanding of nervous disorders of the stomach and intestine. (New York: Harper Collins, p. 163.) If serum 5HT is elevated, there are abnormalities in the bowel that lead to excessive 5HT release. (Gershon 1998).) Serotonin/secretin co-localization and the number of S cells have been found to be markedly altered in the autistic gut. (Gershon, et al., Personal communication 2003.) Secretin has long been recognized as a gut peptide (Bayliss, et al., 1902). The mechanism of pancreatic secretion (J. Physiol. (Lond) 28, 325-353), and more recently as a neuropeptide. (Welch, et al., Secretin: hypothalmamic distribution and hypothesized neuroregulatory role in autism; Cell. Mol. Neurobiol., 2004, 24(2):167-89.)
Recent studies have demonstrated pathology in the gastrointestinal tract of autistic children, including findings of impaired gut/immune system development, altered production of gut/brain peptides, increased intestinal mucosal permeability, and inflammation. (Warren, et al., 1997). Brief report: immunoglobulin: A deficiency in a subset of autistic subjects, J. Autism. Dev. Disord., 27(2): 187-92; Nelson, et al., Neuropeptides and neurotrophins in neonatal blood of children with autism or mental retardation. Ann. Neurol., (2001) 49(5):597-606; Torrente, et al., 2002). Small intestinal enteropathy with epithelial IgG and complement deposition in children with regressive autism. Mol. Psychiatry; 7(3):375-82, 334; White 2003). Intestinal pathophysiology in autism. Exp. Biol. Med. (Maywood) 228(6):639-49)). Other results have highlighted the possibility of homeostatic imbalance in autistic children (Chugani, et al., 1999). Evidence of altered energy metabolism in autistic children; Prog. Neurophsychopharmacol Biol. Psychiatry, 23(4):635-41), and have called attention to the dysregulation of peptide hormones protective of homeostasis. (Nelson, et al., Neuropeptides and neurotrophins in neonatal blood of children with autism or mental retardation; Ann. Neurol. (2001) 49(5):597-606; Hollander, et al., 2003). Oxytocin infusion reduces repetitive behaviors in adults with autistic and Asperger's disorders. Neuropsychopharmacology, 28(1): 193-8; Gerson, et al., Personal communication 2003). Thus, effective clinical or pharmacokinetic intervention for autistic symptoms may require a method that acts simultaneously upon gut/brain and on the associated gut/brain stress axis in order to re-establish homeostasis. (Welch, et al., 2003b) Neurohormonal Resolution of Genetic and Acquired IBD and Secondary Brain Activation in Areas Abnormal in Autism, Neurosci. Abstracts: 33rd Annual Meeting November 8-12.) Furthermore, cerebral metabolic imbalances in autism have been identified via functional imaging. (Haznedar, et al., 2000). Limbic circuitry in patients with autism spectrum disorders studied with positron emission tomography and magnetic resonance imaging. Am. J. Psychiatry, 157(12): 1994 -2001); Naturalistic and/or peptide therapies (Welch 1983a), Retrieval from autism through mother-child holding. In Tinbergen N and EA. Autistic Children—New Hope for a Cure. London and Boston: George, Allen and Unwin; Welch 1983b), Retrieval from autism through mother-child holding therapy. In Call, et al., eds. Frontiers of Infant Psychiatry (1983b) New York: Basic Books; Horvath, et al., 1998). Improved social and language skills after secretin administration in patients with autistic spectrum disorders. J. Assoc. Acad. Minor Phys.; 9(1): 9-15; Hollander, et al., 2003) Oxytocin infusion reduces repetitive behaviors in adults with autistic and Asperger's disorders. Neuropsychopharmacology, 28(1):193-8; Welch, et al., 2003b) Neurohormonal Resolution of Genetic and Acquired IBD and Secondary Brain Activation in Areas Abnormal in Autism, Neurosci. Abstracts: 33rd Annual Meeting November 8-12) may be effective to the extent that they correct such imbalances. Peptide infusions can access the sympathetic ganglia that regulate systemic and cerebral microvasculature (Palmer, Neurochemical coupled actions of transmitter in the microvasculature of the brain, Neurosci. Biobehav. Rev. (1986) Summer; 10(2):79-101) and innervate the pineal gland, the richest source of secretin. (Chariton, et al., Secretin immunoreactivity in rat and pig brain, Peptides (1981) 2 Suppl 1:45-9.) Two studies of peptide treatments, epidermal growth factor enemas and systemic VIP, a member of the secretin family, have demonstrated efficacy in ameliorating IBD in humans and in an animal model, respectively (Farrell, Epidermal growth factor for ulcerative colitis, N. Engl. J. Med. (2003) 349(4):395 -7; Abad, et al., Therapeutic effects of vasoactive intestinal peptide in the trinitrobenzene sulfonic acid mice model of Chrohn's disease, Gastroenterology (2003) 124(4):961-71). However, neither study examined the treatment's effect on the brain.
It remains a major therapeutic challenge to find new effective approaches to improve the diagnosis and treatment of GI and other brain/gut disorders. The etiology of psychiatric disorders such as autism, as well as the link between these psychiatric disorders and gastrointestinal abnormalities remains poorly understood. Accordingly, a need exists for the development of novel therapeutic measures for treating patients with psychiatric and physical illness associated with chronic visceral inflammation.
The rationale for examining peptide treatment for visceral inflammation and its concomitant brain changes emerges from clinical work demonstrating the efficacy of intensive maternal nurturing and from the literature on maternal nurturing in animal models. (Meaney, et al., Effect of neonatal handling on age-related impairments associated with the hippocampus, Science (1988) 239(4841 Pt 1):766-8) Clinical work (Tinbergen, et al., 1983, Welch, 1983a,b, 1989) has shown that autistic spectrum disorders are ameliorated by reinstating components of maternal nurturing, including the establishment of synchronous attunement between mother and child (Welch, 1983a,b, 1987, 1988 a, b, 1989, Welch, et al., 1988). Mother/infant interaction appears to be a powerful stimulus to neuropeptide release (Matthiesen, et al., 2001). In clinical practice, autistic children and adopted orphans with severe maternal deprivation syndromes were treated with Prolonged Mother-Child Embrace, an intervention that reinstates specific components of maternal nurturing (holding, embracing, comforting, licking, talking, feeding). This intervention has been reported to resolve behavioral and visceral symptoms and restore normal development. (Welch, Appendix I: retrieval from autism through mother-child holding therapy In Tinbergen, et al., ‘Autistic’ Children: New Hope for a Cure. George Allen & Unwin, London (1983a.) 322-336; (1983b) Retrieval from autism through mother-child holding therapy, In Call, et al., eds. Frontiers of Infant Psychiatry (1983b) New York: Basic Books; Toward prevention of developmental disorders, Pa Med. (1987) 90(3):47-52; Welch, Mother-child holding therapy and autism, Pa Med. (1988) 91(10):33-8; Welch, Holding Time. New York: Simon and Schuster (1988); Welch (1989) Holding Time: How When Why, Proceedings of the First International Congress of Holding Therapy, Regensberg, Germany; Welch, et al., Outcomes of an intervention to reinstate maternal nurturing among children with behavioral disorders, In prep.) It is hypothesized that the amelioration of symptoms associated with Prolonged Mother-Child Embrace is the result of a testable mechanism involving S and OT up-regulation. (Rominger, et al., Plasma secretin concentrations and gastric pH in healthy subjects and patients with digestive diseases, Dig. Dis. Sci. (1981) 26(7): 591-7; Peterson, et al., Oxytocin selectively increases holding and licking of neonates in preweanling but not postweanling juvenile rats, Behav. Neurosci. (1991) 105(3): 470-7; Matthiesen, et al., Postpartum maternal oxytocin release by newborns: effects of infant hand massage and sucking, Birth, 2001 28(1):13-9; Pedersen, et al., Oxytocin links mothering received, mothering bestowed and adult stress responses, Stress, 2002 5(4): 259-67; Francis, et al., Naturally occurring differences in maternal care are associated with the expression of oxytocin and vasopressin (V1a) receptors: gender differences, J. Neuroendocrinol. (2002) 14(5): 349-53; Welch, et al., Outcomes of an intervention to reinstate maternal nurturing among children with behavioral disorders, In prep.) Brain/gut neuropeptides contribute to developmental neuroregulation of growth, differentiation and regeneration and to the control of hormone release (Houben, et al., 1994), as well as to the resolution of visceral inflammation and brain activation in brain/gut dysregulation models (Welch, et al., 2002b). These studies indicate that maternal nurturing, as well as interventions that effectively replicate it, involves ameliorative mechanisms that stimulate neuropeptide release.
The inventors' propose that some childhood development abnormalities are spectrum disorders of brain/gut dysregulation that can be ameliorated by naturalistic and/or peptide therapy. Recent research has reveled pathology in the gastrointestinal tract of autistic children extending from the esophagus to the colon. This finding has led to investigations of impaired gut/immune system development, altered production of brain/gut peptides, increased intestinal mucosal permeability, and inflammation (Warren, et al., 1997; Nelson, et al., 2001; Torrente, et al., 2002; White 2003). Other evidence has focused attention on the possibility of homeostatic imbalance, such as altered central and peripheral energy metabolism in autistic children (Chugani, et al., 1999), and dysregulation of peptide hormones that protect homeostasis (Nelson, et al., 2001; Hollander, et al., 2003b; Gershon 2003 personal communication). Effective clinical or pharmacokinetic intervention in autistic symptomatology will require a mechanism that acts simultaneously upon the mind/brain/body stress axis to re-establish homeostasis (Welch, et al., 2002b). Metabolic imbalances in autism have been defined via fMRI (Haznedar, et al., 2000). Naturalistic and/or peptide therapies (Welch 1983a,b; Horvath, et al., 1998; Hollander, et al., 2003b; Welch, et al., 2003b) will be effective to the extent that they address such imbalances.
Psychotherapeutic and pharmacologic measures (Langworthy-Lam, et al., 2002; Diggle, et al., 2003), including peptide neurohormone administration, have been attempted in autistic children, with limited outcomes (Horvath, et al., 1998; Lamson 2001; Sandler, et al., 1999; Coniglio, et al., 2001; Dunn-Geier, et al., 2000; Koren 2001; Lightdale, et al., 2001; Owley, et al., 2001; Roberts, et al., 2001; Kern, et al., 2002). Research supports the importance of peptides in treating behavioral and developmental disorders in autistics: at the bedside, through systemic peptide administration (Horvath, et al., 1998; Hollander, et al., 2003b), in clinical studies (Matthiesen, et al., 2001), and in experimental animals, through reinstating components of maternal nurturing, such as feeding, handling, and licking (Francis, et al., 2002; Bredy, et al., 2003). Experimental models show that feeding and handling ameliorate brain pathology resulting from the social-isolation stress of maternal deprivation (Meaney, et al., 1988, 1991; Anisman, et al., 1998). One peptide in particular, secretin, is associated with feeding and handling, a form of controlled restraint (Lauterbach, et al., 1980; Mineo, et al., 1990).
Secretin is a bioactive peptide endogenously and predominantly synthesized by upper intestinal secretin S cells (Bloom, et al., 1974; Miller, et al., 1978; Strauss, et al., 1978; Paquette, et al., 1982; Chang, et al., 1999). It is also synthesized in mice by the pancreas and colon (Lopez, et al., 1995), and by flora that inhabit the gut (Gauthier, et al., 2003). Whether secretin is synthesized by the forebrain is the subject of this study. Secretin belongs to the secretin/vasoactive intestinal peptide (VIP)/glucagon receptor family with actions at high and low-affinity secretin receptors (Ichihara, et al., 1983). It is a twenty-seven amino acid peptide and an enterogastrone (Jin, et al., 1994; Li, et al., 1998). Secretin receptors couple to G-proteins that stimulate adenylate cyclase and, in turn, lead to the production of cyclic adenosine monophosphate (cAMP) and the stimulation of associated second messenger systems (Harmar 2001). Secretin receptors concentrate in brain regions (Itoh, et al., 1991; Ohta, et al., 1992) that are responsive to intracerebroventricular (i.c.v.) administration of the secretin peptide (Welch, et al., 2002a,b, 2003a), brain regions that are also sites of pathology in autism (Bauman, et al. 1985; Haznedar, et al., 2000; Ogai, et al., 2003; Schultz, et al., 2003).
Secretin's peripheral role as a gastric hormone has long been established (Bayliss and Starling 1902). Less is known about the central actions of secretin. Secretin regulates the central and peripheral stress axes via neurohumoral mechanisms (Ruggiero, et al., 2003; Welch, et al., 2002a,b, 2003a,b) that involve interactions with other signaling systems acting at the level of the hypothalamus, such as secretin/angiotensin (Walker, et al., 1999) and secretin/dopamine (Fuxe, et al., 1979). Secretin functions to modulate HPA stress axis output, and, in contrast to VIP, increases norepinephrine and dopamine turnover in the hypothalamus and median eminence (Fuxe, et al., 1979). Assays finding positive secretin bioactivity, radioimmunoreactivity, or secretin precursor mRNA expression indicated high secretin levels in the hypothalamus and hypophysis, with the preponderance of evidence suggesting that the hypothalamus is a site of origin of endogenous secretin (Fuxe, et al., 1979; Mutt, et al., 1979; Charleton, et al., 1981; O'Donohue, et al., 1981; Samson, et al., 1984; Chang, et al., 1985; Itoh, et al., 1991; Ohta, et al., 1992; Nussdorfer, et al., 2000). Another study assessing the presence of secretin in the rat brain and gut did not find central expression of the bioactive peptide (Kopin, et al., 1990). These studies, however, lacked the single cell resolution needed to precisely delineate the organization of a presumptive secretinergic system. Recently, studies found secretin immunoreactivity in the brain stem and cerebellum (Yung, et al., 2001; Koves, et al., 2002; Ng, et al., 2002), but not in the forebrain. According to Ng, “secretin is only present at detectable levels in the brainstem and cerebellum,” although unpublished data suggest “the presence of secretin-producing cells in the cerebral cortex” (Ng, et al., 2002).
The inventors previously tested the hypothesis that secretin regulates stress response patterns via endogenous synthesis along the hypothalamic stress axis. Central secretin administration, i.c.v., activates the area postrema, nucleus of solitary tract (NTS) and its terminal fields, including parabrachial complex, amygdala, and hypothalamus. In addition, secretin activates the visceral thalamus and its insula/orbital and medial prefrontal cortical projection fields, which regulate visceral reflex networks overlapping areas of pathology in autism (Welch, et al., 2002a,b, 2003a). Corroborating some of these results was a report focused on the effects of secretin-induced c-fos gene expression in the amygdala of rats (Goulet, et al., 2003). In a subsequent study, long-term systemic administration of bioactive peptides, including trials with secretin, was found to resolve inflammatory bowel lesions and stress-related effects on specific CNS regions that corresponded to sites of pathology in autism (Welch, et al., 2003b). Systemic exogenous secretin was found to reestablish communicative and affiliative interactions in autistic children with gastrointestinal abnormalities (Horvath, et al., 1998), an observation supported by Lamson (Lamson, et al., 2001). This interest in secretin has led to a current investigative effort to replicate Horvath's novel peptide therapy (Wheeler 2003).
Before the present invention, there was a study demonstrating secretinergic neurons in the forebrain with single cell resolution. The inventors sought to determine whether secretin is synthesized specifically in the forebrain, and whether its specificity and distribution patterns might predict possible interactions of secretin with other hormones involved in stress adaptation. The inventors also investigated whether secretin is synthesized on demand in distinct areas of the HPA stress axis of rats. The central distribution of secretin immunoreactivity in adult male Sprague-Dawley rats was mapped to single-cell resolution using immunocytochemistry.
Although secretin has been identified primarily as a gut hormone, the inventors demonstrated secretinergic immunoreactivity in the hypothalamus of a rat. Secretin also has been shown to have regulatory effects on other organ systems, including the immune system, the central nervous system, the endocrine system, and the respiratory and cardiovascular systems. Van Tol, et al., found that secretin plays a role in the regulation of cellular cytotoxicity against tumor cells. Several studies have reported secretin immunoreactivity in widespread areas of the central nervous system (Chang, et al., 1985; Mutt, et al., 1979; O'Donohue, et al., 1981). Many of the studies have focused particularly on the hypothalamus of various species (Chang, et al., 1985; Charlton, et al., 1981; Mutt, et al., 1979; O'Donohue, et al., 1981; Samson, et al., 1984). Though Kopin, et al. (1990) failed to detect secretin mRNA in the central nervous system (CNS), Itoh, et al., 1991 and Ohta, et al. 1992, reported CNS expression of an mRNA precursor to secretin in the brainstem, thalamus, hypothalamus, and cerebral cortex. In a later study by Nozaki, et al., 2002, secretin was found to bind with specificity and high affinity to the nucleus of the solitary tract, and other regions in the brainstem, thalamus, hypothalamus, and cerebral cortex.
Secretin has long been thought to be a central neuromodulator, prompting several studies examining the actions of secretin injected into the cerebroventricular system: Charlton, et al., 1981, found that intracerebroventricular (i.c.v.) injection of secretin in rats significantly increased defecation, decreased novel-object approaches and open-field locomotor activity, and altered respiration. Weick, et al., 1992, showed that i.c.v. secretin administration inhibited pulsatile luteinizing hormone secretion in the ovariectomized rat. It is conceivable that secretin is synthesized endogenously in the central nervous system (Fuxe, et al., 1979; Itoh, et al., 1991; O'Donohue, et al., 1981; Ohta, et al., 1992). In the cardiovascular system, Gunnes, et al., 1983, found in human subjects that secretin has both iontropic and vasodilating effects. Kitani, et al., 1978, found that secretin increased cardiac output distribution to the stomach, small intestine, and pancreas in rats. In another study, i.c.v. secretin injection induced hypothermia and elevated blood pressure without effects on heart rate (Shido, et al., 1989), although the central sites of action were not identified.
Taken together, these data suggest that secretin is a regulatory hormone with peripheral and central mechanisms of action on multiple organ systems. Although one hypothesized mechanism of secretin is its well-established role in regulating gut function, secretin's regulatory role in other organ systems may underlie some of its ameliorative, though short-lived, actions in autistic children (Horvath, et al., 1998). Earlier studies have localized secretin and its presumptive receptor binding sites in viscerolimbic brain regions involved in central autonomic regulation (Itoh, et al., 1991; Nozaki, et al., 2002; Ohta, et al., 1992). Before the present invention, no study had examined the effects of i.c.v. administration of secretin on visceral brain regions that may be differentially activated.
Oxytocin is a nine amino acid peptide that is synthesized in hypothalamic neurons and transported down axons of the posterior pituitary for secretion into the blood. Oxytocin's physiological roles include stimulation of uterine contraction during childbirth, establishment of maternal behavior, and to cause milk ejection from the breast by contraction of the myoepithelial cells in response to suckling (let down reflex). In males, oxytocin is involved in facilitating sperm transport within the male reproductive system. Acute stress can inhibit oxytocin release. For example, oxytocin neurons are repressed by catecholamines, which are released from the adrenal gland in response to stress. Surprisingly, the inventors of the present invention have discovered that localizations of secretin overlap with those of oxytocin.
The inventors have shown for the first time that peptide therapy, particularly therapy utilizing co-administration of secretin and oxytocin, provides a simultaneous resolution of gut and brain disorders. Gut and brain areas affected by co-administration of secretin and oxytocin overlap those affected in autism, including: thalamus amygdale, HF, Cingulate orbital frontal insula, and PFC.